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P14(ARF) (p19(ARF) in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14(ARF) to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14(ARF) is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3sigma or both, we further investigated the cooperative effect of p21 and 14-3-3sigma on cell cycle regulation and apoptosis induction by p14(ARF). In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3sigma-deficient cells, we show here that the expression of p14(ARF) triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3sigma. The activation of the intrinsic mitochondrial apoptosis pathway by p14(ARF) was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3sigma/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14(ARF) as compared to wild-type cells or cells lacking either gene alone. Notably, p14(ARF)-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14(ARF) impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14(ARF)-induced mitochondrial apoptosis.

Among the members of the Bcl-2 family, the multidomain proteins Bax and Bak are crucial for the activation of mitochondria. However, it is still unclear whether they act in a unique and distinct manner or whether they exhibit redundant functions. To systematically investigate their activation on a single-cell level, we established MCF-7 cell lines stably expressing GFP-fusion variants of these proteins. We found that MCF-7/GFP-Bak cells showed an increased sensitivity to apoptosis induction by staurosporine, actinomycin D, TRAIL and overexpression of Puma compared to GFP-Bax-expressing cells. Independently of the death stimulus used, oligomerization of endogenous and exogenous Bak was mostly detected prior to an activation of Bax, whereas cells displaying oligomerized Bax in the absence of Bak clusters were not observed. In addition, activation of Bax but not Bak was attenuated by a caspase inhibitor. Consistent with this, caspase-3-deficient MCF-7 cells displayed a significantly reduced activation of endogenous Bax than caspase-3-proficient MCF-7 cells. Thus, our data strongly suggest that diverse apoptotic stimuli preferentially engage the Bak pathway, whereas the triggering of Bax occurs, at least partially, downstream of mitochondrial caspase activation, most likely constituting a positive feedback loop for the amplification of the death signal.

Renal cell carcinoma (RCC) is resistant to chemotherapy, and this resistance is mirrored by a high apoptosis resistance of many RCC lines in vitro. Here, we report the loss of the pro-apoptotic BH3-only protein Bim in a large part of clinical RCC cases and provide evidence for a functional relevance of this loss. Immunohistochemistry of clear cell renal cell carcinoma cases and corresponding normal kidney showed strong Bim reactivity in renal tubules of all cases but loss of Bim in 35 of 45 RCC samples. Out of nine RCC cell lines investigated, six showed strongly diminished or undetectable levels of Bim protein by western blotting. Four RCC lines of varying apoptosis sensitivity were analysed further. Bcl-2, Bcl-x(L), Mcl-1, Bax and Bak expression did not correlate with apoptosis sensitivity. All cell lines underwent apoptosis upon forced expression of Bax and Bim, suggesting an upstream difference. In all four lines, adriamycin induced p53 but not its targets Puma or Noxa. However, apoptosis sensitivity correlated with levels of Bim protein. Bim siRNA reduced apoptosis sensitivity in a susceptible cell line. Furthermore, inhibition of histone deacetylation restored Bim expression in cell lines. These data suggest that Bim has a function as a tumor suppressor in RCC.

In contrast to the initial notion that the biological activity of p14(ARF) strictly depends on a functional mdm-2/p53 signaling axis, we recently demonstrated that p14(ARF) mediates apoptosis in a p53/Bax-independent manner. Here, we show that p14(ARF) induces breakdown of the mitochondrial membrane potential and cytochrome c release before triggering caspase-9- and caspase-3/7-like activities in p53/Bax-deficient DU145 prostate cancer cells expressing wild-type Bak. Re-expression of Bax in these cells failed to further enhance p14(ARF)-induced apoptosis, suggesting that p14(ARF)-induced apoptosis primarily depends on Bak but not Bax in these cells. To further define the role of Bak and Bax in p14(ARF)-induced mitochondrial apoptosis, we employed short interference RNA for the knockdown of bak in isogeneic, p53 wild-type HCT116 colon cancer cells either proficient or deficient for Bax. There, combined loss of Bax and Bak attenuated p14(ARF)-induced apoptosis whereas single loss of Bax or Bak was only marginally effective, as in the case of DU145. Notably, HCT116 cells deficient for Bax and Bak failed to release cytochrome c and showed attenuated activation of caspase-9 (LEHDase) and caspase-3/caspase-7 (DEVDase) upon p14(ARF) expression. These data indicate that p14(ARF) triggers apoptosis via a Bax/Bak-dependent pathway in p53-proficient HCT116, whereas Bax is dispensable in p53-deficient DU145 cells. Nevertheless, a substantial proportion of p14(ARF)-induced cell death proceeds in a Bax/Bak-independent manner. This is also the case for inhibition of clonogenic growth that occurs, at least in part, through an entirely Bax/Bak-independent mechanism.

The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk/Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk/Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk/Bik only weakly at the mRNA and protein level. Conditional expression of Nbk/Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk/Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk/Bik expression. By investigating the mechanism of Nbk/Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk/Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk/Bik.

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

The mitochondrial apoptosis pathway mediates cell death through the release of various pro-apoptotic factors including cytochrome c and Smac, the second mitochondrial activator of caspases, into the cytosol. Smac was shown previously to inhibit IAP proteins and to facilitate initiation of the caspase cascade upon cytochrome c release. To investigate Smac function during apoptosis and to explore Smac as an experimental cancer therapeutic, we constructed an expression system based on a single adenoviral vector containing Smac under control of the Tet-off system supplied in cis. Conditional expression of Smac induced apoptosis in human HCT116 and DU145 carcinoma cells regardless of the loss of Bax or overexpression of Bcl-x(L). Nevertheless, apoptosis induced by Smac was associated with cytochrome c release and breakdown of the mitochondrial membrane potential. This indicates that Smac acts independently of Bax and Bcl-x(L) during initiation of apoptosis and triggers a positive feedback loop that results in Bax/Bcl-x(L)-independent activation of mitochondria. In caspase-proficient cells, Smac-induced apoptosis could be inhibited partially by cell-permeable LEHD (caspase-9 inhibitor) and DEVD (caspase-3 inhibitor) peptides. Furthermore, loss of caspase-3 expression in MCF-7 cells carrying a caspase-3 null mutation completely abrogated the sensitivity for Smac-induced apoptotic or nonapoptotic, necrosis-like cell death, while re-expression of caspase-3 conferred sensitivity. Altogether, caspase-3 but not caspase-9 activation was necessary for execution of Smac-induced cell death. Notably, Smac did not induce caspase-9 processing in the absence of caspase-3. Thus, caspase-9 processing occurs secondary to caspase-3 activation during Smac-induced apoptosis. Altogether, Smac is capable of circumventing defects in mitochondrial apoptosis signaling such as loss of Bax or overexpression of Bcl-x(L) that are frequently observed in tumor cells resistant to anticancer therapy. Consequently, Smac appears to be a promising therapeutic target in anticancer treatment.

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.

Knowledge of the type of biological reaction to chemotherapy is a prerequisite for its rational enhancement. We previously showed that irinotecan-induced DNA damage triggers in the HCT116p53(wt) colon carcinoma cell line a long-term cell cycle arrest and in HCT116p53(-/-) cells apoptosis (Magrini et al., 2002). To compare the contribution of long-term cell cycle arrest and that of apoptosis to inhibition of cell proliferation after irinotecan-induced DNA damage, we used this isogenic system as well as the cell lines LS174T (p53(wt)) and HT-29 (p53(mut)). Both p53(wt) cell lines responded to damage by undergoing a long-term tetraploid G1 arrest, whereas the p53(mut) cell lines underwent apoptosis. Cell cycle arrest as well as apoptosis caused a similar delay in cell proliferation. Irinotecan treatment also induced in mouse tumours derived from the p53(wt) cell lines a tetraploid G1 arrest and in those derived from the p53-deficient cell lines a transient G2/M arrest and apoptosis. The delay of tumour growth was in the same range in both groups, that is, arrest- and apoptosis-mediated tumour growth inhibition was comparable. In conclusion, cell cycle arrest as well as apoptosis may be equipotent mechanisms mediating the chemotherapeutic effects of irinotecan.